Abstract Many diseases can produce signatures of their infiltration by spilling markers into peripheral blood. For example, in the case of epithelial-based cancers, markers found in blood include circulating tumor cells (CTCs), exosomes and cell-free DNA (cfDNA). Thus, blood-based diagnostics has the potential to identify early cancer, guide personalized treatment decisions and monitor recurrence and treatment efficacy. This situation is true in other important diseases, such as stroke and infectious diseases. In spite of the compelling attributes associated with circulating markers, clinical decisions are for the most part predicated on imaging technologies. Unfortunately, most settings have limited access to current imaging-based technologies. Further, these technologies often generate false-positives, are limited to a single dimension and are not cost-effective for highly accurate early detection. To solve this urgent and unmet medical need, TR&D Project 2 will develop a suite of isothermal enzymatic reactions to enable comprehensive and multi-dimensional molecular profiling of DNA mutations and methylation, gene copy number variations, and mRNA/miRNA expression. A new paradigm for blood-based detection termed ?circulating marker load? analogous to viral load is envisioned. Input whole blood samples are processed using the innovative technologies emanating from TR&D Project 1 to isolate biological cells, cfDNA, and exosomes even if they are rare events. TR&D Project 2 will develop compelling molecular processing strategies taking advantage of a chamber array that consists of 50 ? 50,000 chambers with 8 pixels per chamber and each pixel comprised of 288 pillars for a total of 400 ? 400,000 pixels. DNA or copies of RNA targets will be tailed with TTP nucleotides using Terminal deoxynucleotidyl Transferase and captured via base pairing to poly-dA30 primers covalently attached to the pillars. Bst polymerase is used to linearly extend the poly-dA30 primers from original DNA or cDNA. Promoter methylation status at defined CGCG sites will be determined by replicating genomic fragments in the presence of BstU1 endonuclease. Linear extension on the solid support enables solid-phase ligase detection reactions (spLDR) to identify and enumerate the desired mutation, methylation status or RNA sequences. The innovative feature of this project is that the input biological sample, which may contain up to 400 billion molecules per mL, is distributed and immobilized to an array of pixels so that biological errors associated with the enzymatic reaction can be obviated by re-interrogating specific pixels. TR&D Project 1 modules will provide high purity molecules, even when in low abundance, isolated from whole blood to this TR&D Project, which in turn will provide spLDR products for nano-scale flight tube detection in TR&D Project 3. Collaborative Projects will be used to validate the clinical utility of the proposed assays and the associated hardware emanating from all TR&D Projects including the system emanating from TR&D Project 4.